Self assembled patterning using patterned hydrophobic surfaces

ABSTRACT

Embodiments provided herewith are directed to self-assembled methods of preparing a patterned surface for sequencing applications including, for example, a patterned flow cell or a patterned surface for digital fluidic devices. The methods utilize photolithography to create a patterned surface with a plurality of microscale or nanoscale contours, separated by hydrophobic interstitial regions, without the need of oxygen plasma treatment during the photolithography process. In addition, the methods avoid the use of any chemical or mechanical polishing steps after the deposition of a gel material to the contours.

INCORPORATION BY REFERENCE TO PRIORITY APPLICATIONS

The present application is a continuation of U.S. application Ser. No.16/096,669, filed Oct. 25, 2018, to be issued as U.S. Pat. No.10,900,076, which is a U.S. national phase application under § 371 ofInternational Appl. No. PCT/US2017/033169, filed May 17, 2017, whichclaims the benefit of priority to U.S. Provisional Appl. No. 62/338,394,filed May 18, 2016, each of which is hereby incorporated by reference inits entirety.

BACKGROUND

In general, the present disclosure relates to the fields ofphotolithography patterning processes to produce micro- ornano-patterned surfaces for polynucleotide sequencing applications. Morespecifically, the present application relates to self-assembled methodsof preparing patterned surfaces for flow cells or digital fluidicdevices.

SUMMARY

Flow cells are devices that allow fluid flow through channels or wellswithin a substrate. Patterned flow cells that are useful in nucleic acidanalysis methods include discrete wells having an active surface withininert interstitial regions. The surface of the flow cell is normallyfabricated using the following steps: (1) wells are initially etchedinto a uniform substrate; (2) the wells and the interstitial regions arefunctionalized with a silane and a gel material; (3) excess gel materialcovering the interstitial regions is removed via a polishing process;and (4) the gel material in the wells is then grafted with singlestranded primer DNA to provide a flow cell surface for the downstreamsequencing applications. In this case, some of the gel material iswasted in the polishing step of the fabrication workflow. In addition,the surface energy of the interstitial regions largely depends on thestarting substrate.

Embodiments relate to self-assembled methods of preparing a patternedsurface for sequencing applications. The patterned surface may include,for example, a patterned flow cell or a patterned surface for a digitalfluidic device. In some embodiments, the methods utilizephotolithography to create a patterned surface with a plurality ofmicroscale or nanoscale contours separated by hydrophobic interstitialregions while eliminating the need for oxygen plasma surface or othertreatment before deposition of a photoresist. In addition, someembodiments avoid the use of chemical or mechanical polishing stepsafter the deposition of a gel material over the contours.

Some embodiments described herein are related to methods of preparing apatterned surface with gel-coated contours by: providing a solid supportcomprising a surface, the surface comprising a continuous hydrophobiccoating layer; disposing a photoresist on the hydrophobic coating layerof the solid support to cover the hydrophobic coating layer; andpatterning the photoresist layer by photolithography (or other suitablemethods known in the art and/or described herein) to form micro-scale ornano-scale contours on the surface; and depositing a layer of a gelmaterial within the micro-scale or nano-scale contours, wherein the gelmaterial is capable of covalently bonding to oligonucleotides. In someembodiments, the micro-scale or nano-scale contours are formed byetching off portions of the hydrophobic coating layer. In someembodiments, the micro-scale or nano-scale contours are separated fromeach other by hydrophobic interstitial regions comprising thehydrophobic coating layer. In some embodiments, at least a portion ofthe micro-scale or nano-scale contours are free of hydrophobic coating.In some embodiments, the methods do not require a plasma surfacemodification treatment (e.g., descum or oxygen plasma treatment, coronatreatment, heating, chemical or liquid activation, or other treatmentused to increase surface energy and improve bonding characteristics) ofthe surface prior to disposing the photoresist. In certain embodiments,the contours are wells.

Some embodiments described herein are related to methods of preparing apatterned surface for analytic applications, the methods include:providing a solid support comprising a surface, the surface comprising acontinuous hydrophobic coating layer; disposing a photoresist on thehydrophobic coating layer of the solid support to cover the hydrophobiccoating layer; patterning the photoresist layer by photolithography (orother suitable methods) to form micro-scale or nano-scale contours onthe surface separated by hydrophobic interstitial regions; removing thephotoresist; and applying a layer of binding material, such as a silanelayer, to the surface to cover at least a portion of the contours and aportion of the hydrophobic interstitial regions. In some embodiments,the methods further include covalently attaching a gel material to thelayer of binding material, such as silane. In some embodiments, themethods further include covalently attaching an oligonucleotide to thegel material. In some embodiments, the methods further includenon-covalently attaching a gel material to the layer of bindingmaterial, such as silane. In some embodiments, the methods compriseapplying a layer of gel material to the binding material layer or layerof silane.

Some embodiments described herein are related to methods of preparing apatterned surface for analytic applications, the methods include:providing a solid support comprising a surface, the surface comprising acontinuous hydrophobic coating layer; disposing a photoresist on thehydrophobic coating layer of the solid support to cover the hydrophobiccoating layer; patterning the photoresist layer by photolithography (orother suitable methods) to form micro-scale or nano-scale contours onthe surface separated by hydrophobic interstitial regions; applying alayer of silane to the surface to cover at least a portion of thecontours and a portion of the hydrophobic interstitial regions; andcovalently attaching a gel material to the binding material layer orlayer of silane. In some embodiments, the methods further includeremoving the photoresist to expose the hydrophobic layer in hydrophobicinterstitial regions.

In some embodiments, the binding material affixes, covalently ornon-covalently, the gel material to the hydrophobic coating layer and/orthe solid support. In some embodiments, the gel material is covalentlybound to the binding material, e.g., silane.

Some embodiments described herein are related to methods of preparing anarray of polynucleotides, the methods include providing a solid supportcomprising a patterned surface, the surface comprising microscale and/ornanoscale contours coated with a gel material that is capable ofcovalently bonding to oligonucleotides, the surface is prepared by anyof the methods described herein; and covalently attaching a plurality offirst oligonucleotides and a plurality of second oligonucleotides to thegel material.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing three types of perfluorinatedpolymers (CYTOP-A, CYTOP-M and CYTOP-S) that may be used to create thehydrophobic interstitial regions on the patterned surface of asubstrate.

FIG. 2A is a cross section partial view of a patterned surface of aglass substrate including a plurality of wells and hydrophobicinterstitial regions.

FIG. 2B is a magnified view of FIG. 2A showing three layers of materialson top of a glass slide.

FIG. 2C are fluorescence images of three grafted glass surfaces from aTyphoon instrument.

FIG. 3 illustrates the contact angles of CYTOP-M and CYTOP-S treatedglass surface compared to the control surface before and after chemicaltreatment.

FIG. 4 illustrates a cross-section view of a typical workflow forcreating a patterned surface.

FIG. 5 illustrates one embodiment of an improved workflow for creating apatterned surface.

FIGS. 6A and 6B are fluorescent images of a patterned device surfacewith 14 μm microwells.

FIG. 6C is a fluorescent image of patterned DNA clusters in 700 nmdiameter microwells.

FIG. 7 illustrates one embodiment of an improved direct patterningworkflow for creating a patterned surface.

FIG. 8A illustrates one embodiment of an improved patterning workflowfor creating a patterned surface using lift-off method.

FIG. 8B illustrate a fluorescent image of patterned PAZAM and DNAclusters in 14 diameter μm microwell structures using the lift-offworkflow exemplified in FIG. 8A.

FIG. 9A illustrates an example of a liquid phase reflow process.

FIG. 9B illustrates an example of a vapor phase reflow process.

FIG. 10A is an optical microscopy image of PAZAM coated CYTOP-Snanowells (diameter of 700 nm to 1.1 μm) after O₂ plasma treatment.

FIG. 10B is an optical microscopy image of PAZAM coated CYTOP-Snanowells in FIG. 10A after liquid phase solvent reflow.

FIG. 10C is a fluorescence image of the PAZAM coated CYTOP-S nanowellsof FIG. 10B after the wetting/dewetting of an aqueous droplet withfluorescent die.

FIG. 11A depicts DNA cluster patterning in sub-micron sized wells. Thebright spots are fluorescent dye labeled DNA clusters. In the “t”-shapedarea, the material is SiO₂ without patterning, and the clusters arerandomly distributed. The image was generated using the topologydepicted in FIG. 11B.

FIG. 11B depicts a cross-sectional illustration for the patternedsubstrate. The wells have a 0.7 micron diameter arrayed with a 1.75micro pitch.

FIG. 11C shows the patterned substrate assembly in a MiSeq® flowcellformat to facilitate the exchange of bio-reagents. The picture wasgenerated using the topology depicted in FIG. 11B.

FIGS. 12A and 12B show that DNA cluster size and intensity are tunablewith different well sizes. FIG. 12A shows SYTOX®-dyed clusters grown in0.7 micron diameter wells and 1.75 micron pitch with the surface layersas depicted in FIG. 11B. FIG. 12B shows SYTOX®-dyed clusters grown in0.9 micron diameter wells and 1.75 micron pitch.

FIGS. 13A-13C show the sequencing results from the CYTOP patternedsurface (well diameter 0.7 micron; pitch 1.75 micron). FIG. 13A is afirst base image showing super-imposed C and T channels and circularfiducial indicating patterning of clusters within CYTOP nanowells,obtained using a polish-free patterning method. FIG. 13B shows themismatch rate (error rate) for read 1 and read 2 of this run, each 150cycles. The achieved error rate is similar to that expected from MiSeqruns (approx. 2% at the end of 150 cycles). This figure alsodemonstrates that paired-end turn-around is compatible with thepolish-free patterning method. FIG. 13C shows the q-score (qualityscore) of the reads as a function of cycle for the run, while provides ameasure of quality of the base output. CYTOP patterning is shown to becompatible with sequencing chemistry and is robust, slowing thousands offlow exchanges to finish 2×150 bps sequencing. Details for theseexperiments are described in Example 3.

DETAILED DESCRIPTION

Embodiments relate to methods of preparing a patterned surface that canbe used in analysis and synthesis of analytes of interest such asbiological components including, but not limited to, cells, subcellularcomponents, and molecules. Exemplary biological molecules include, butare not limited to nucleic acids, oligonucleotides, nucleotides, aminoacids, peptides, proteins, polysaccharides, sugars, metabolites, enzymecofactors, and the like. Particularly useful analytical processes forwhich the patterned surfaces can be used include, for example, nucleicacid sequencing applications. In one embodiment, the patterned surfaceis part of a flow cell or electrowetting fluidic device. In someembodiments are used nanofabrication techniques, such as photoetching,photoengraving, or photolithography, or other patterning methods such ase-beam lithography, nano imprint lithography, nano-stamping, or directablation, to create the patterned surface with a plurality of microscaleor nanoscale contours, separated by hydrophobic interstitial regions.Where photolithography is mentioned herein as a patterning technique,these other methods may also be used to pattern the surfaces. Thepatterned surface may be manufactured without the need of oxygen plasmatreatment of the substrate surface prior to photolithography. A gelmaterial can be deposited on the surface and differentialhydrophobic/hydrophilic characteristics of the contours and interstitialregions on the surface can be exploited to conveniently remove gelmaterial from some regions of the surface while retaining gel materialat desired features. For example, gel material can be retained atsilanated wells and removed from hydrophobic interstitial regions aroundthe wells. Such embodiments can be particularly advantageous by avoidingthe use of harsh chemical or mechanical polishing steps to remove gelmaterial from interstitial regions after the deposition of the gelmaterial over the surface. In some embodiments, the hydrophobicinterstitial regions comprise a perfluorinated polymer such as CYTOP-S.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Surface Preparations

Some embodiments described herein are related to methods of preparing apatterned surface that is configured to bind analytes, such as nucleicacid molecules, in predetermined positions, and are related to thepatterned surfaces. For example, the patterned surface may have regions,or contours, that have differential hydrophobic and hydrophiliccharacteristics. This can allow surface chemistries to be differentiallyapplied to the surface. For example, contours, wells, or other featuresformed on a surface can be treated to contain reactive silanes and/or agel material that is absent from interstitial regions that separate thecontours, wells, or other features from each other. In one embodiment,the contours or wells are coated with a gel or similar polymericmaterial that is capable of binding to, or is bound to, analytes such asnucleic acid molecules. In this embodiment, the patterned surface can becreated by starting with a solid support comprising a surface having acontinuous hydrophobic coating layer. A layer of photoresist can then bedeposited onto the hydrophobic coating layer of the solid support tocover the hydrophobic coating layer. The photoresist layer can then bepatterned by photolithography using a photomask comprising a pluralityof micro-scale and/or nano-scale patterns such that the micro-scale ornano-scale patterns are transferred to the surface to form micro-scaleor nano-scale contours on the hydrophobic coating layer. A layer of agel material can then be deposited within the micro-scale or nano-scalecontours, wherein the gel material is capable of covalently bonding tooligonucleotides.

During photolithography, photoresist is exposed to a pattern of light(for example, UV light) by using a patterned photomask. The exposure tolight causes a chemical change that allows the portion of thephotoresist that is exposed to be removed by a developer solution toexpose patches of the underlying hydrophobic coating layer. After thephotoresist is developed (for example, by following a standard recipefor the specific photoresist product used in the process), micro-scaleor nano-scale contours are formed by etching off portions of the exposedhydrophobic coating layer on the surface. In some embodiments, themicro-scale or nano-scale contours are separated from each other byhydrophobic interstitial regions comprising the hydrophobic coatinglayer. In some embodiments, at least a portion of the micro-scale ornano-scale contours are free of hydrophobic coating. In someembodiments, the contours comprise depressions, such as channels orwells (for example, microwells or nanowells). In another embodiment, thecontours comprise protrusions, such as ridges, posts, or cones (forexample, nanoposts or nanocones).

In some embodiments of the methods described herein, the hydrophobiccoating layer comprises a fluorinated polymer, a perfluorinated polymer,or a silicon polymer, or a mixture thereof. The polymer backbone may becarbon or silicon, or a combination thereof. In some embodiments, thefluorinated polymer is, for example, an amorphous fluoropolymer(optionally 2,3-linked perfluorinated THF monomers, optionally withpendant functional groups such as carboxyl, silylated amide, ortrifluoromethyl termini, e.g., CYTOP-M, CYTOP-S, CYTOP-A, see FIG. 1), apolytetrafluoroethylene (such as Teflon), parylen (e.g., grades A, F,HT), a fluorinated hydrocarbon, a fluoroacrylic copolymer (such asCytonix Fluoropel), a fluorosilane, or a plasma-deposited fluorocarbon.In some embodiments, the silicon polymer is polydimethylsiloxane or asiloxane. In some embodiments, the hydrophobic coating layer comprises aperfluorinated polymer. In some particular embodiments, theperfluorinated polymer is selected from CYTOP-M, CYTOP-S, or CYTOP-A. Inone embodiment, the perfluorinated polymer comprises or is CYTOP-S. Inanother embodiment, the perfluorinated polymer comprises CYTOP-M. Inanother embodiments, the perfluorinated polymer is a mixture of CYTOP-Sand CYTOP-A. In some embodiments, the hydrophobic coating layer is indirect contact with the surface. The direct contact may be via covalentor non-covalent bonding. In some other embodiments, the hydrophobiccoating layer is in contact with the surface via a first adhesionpromoting layer. In one example, the first adhesion promoting layercomprises a functionalized silane or adhesion promoter; an exemplaryfirst adhesion layer comprises CYTOP-A, (3-aminopropyl)trimethoxysilane(APTMS), or (3-aminopropyl)triethyoxysilane (APTES), or combinationsthereof.

To dispose the photoresist on the hydrophobic coating layer forphotolithography often requires some degree of match in surface energybetween the hydrophobic coating layer and the photoresist layer to bedeposited above it. In some embodiments, the hydrophobic coating layeris prepared for photoresist application using an oxygen plasmatreatment. In some instances, the oxygen plasma treatment (such asdescum treatment) may damage the chemical structure of the hydrophobiccoating layer. The present methods remove or reduce the need for thepre-treatment of the surface prior to disposing the photoresist byoffering two alternative processes.

In one alternative, a photoresist may be used such that it is in directcontact with the hydrophobic coating layer without oxygen plasmapre-treatment. In some embodiments, the photoresist is a positivephotoresist. In other embodiments, the photoresist is a negativephotoresist. In some embodiments, the photoresist is selected fromShipley S1800™ series photoresists, for example, Shipley S1818(MICROPOSIT™ S1818™) and Shipley S1805 (MICROPOSIT™ S1805™).

In another alternative, an adhesion promoting layer may be used toreduce the surface energy mismatch between the hydrophobic coating layerand the photoresist layer. In some embodiments, the adhesion promotinglayer comprises a fluorinated surfactant. In some such embodiments, thefluorinated surfactant is selected from Surflon S-651, Novec FC-4430,Novec FC-4432, Novec FC-4434, Novec FC-5210, Zonyl FSN-100, ZonylFS-300, Zonyl FS-500, Capstone FS-10, Capstone FS-30, Capstone FS-60,Capstone FS-61, Capstone FS-63, Capstone FS-64, or Capstone FS-65, orcombinations thereof. In one embodiment, the fluorinated surfactantcomprises Surflon S-651.

Direct Gel Patterning

Some embodiments described herein are related to methods of preparing apatterned surface for analytic applications by a direct gel patterningmethod. In the direct gel patterning method, a solid support having asurface is provided with a continuous hydrophobic coating layer. Aphotoresist is disposed onto the hydrophobic coating layer. Thephotoresist layer is then patterned by photolithography using aphotomask comprising a plurality of micro-scale or nano-scale patternssuch that the micro-scale or nano-scale patterns are transferred to thesurface to form micro-scale or nano-scale contours on the surfaceseparated by hydrophobic interstitial regions. In some embodiments, themicro-scale or nano-scale contours are formed by etching off portions ofthe hydrophobic coating layer and the micro-scale or nano-scale contoursare separated from each other by hydrophobic interstitial regionscomprising the hydrophobic coating layer. The photoresist is thenremoved and a layer of silane is applied to the surface to cover atleast a portion of the contours and a portion of the hydrophobicinterstitial regions. A gel is then added to the surface. Nucleic acidscan be attached to the gel before, after, or during to the attachment ofthe gel to the surface. These methods are also known as directpatterning methods.

In the direct patterning methods described herein, the remainingphotoresist that is not exposed to light need not undergo any chemicalchange and can remain on the hydrophobic interstitial regions after thedeveloping process. The remaining photoresist may be removed by variousreagents, depending on the type of photoresist used. For example,photoresist lift-off resist (LOR) may be removed by MICROCHEM RemoverPG. In some embodiments, the remaining photoresist is removed byacetone, for example, by sonication in acetone solution. The secondadhesion promoting layer may be removed with the photoresist at the sametime. Alternatively, the second adhesion promoting layer is removedsubsequent to the photoresist using a different removal reagent. Afterthe removal of the remaining photoresist that covers the hydrophobicinterstitial regions, the hydrophobic coating layer that is exposed canbe subjected to subsequent silane deposition.

In some embodiments, a gel material is covalently attached to the layerof silane. In some embodiments, the methods further include curing thegel material. The curing process may be done at various conditions,depending on the type of gel material used, as understood by one ofordinary skill in the art. In some embodiments, the curing is done byincubating the gel material deposited on the silane layer in an oven. Insome further embodiments, the methods further include removing excessgel material such that the hydrophobic interstitial regions aresubstantially free of the gel material. In some embodiments, the removalof the gel material can be simply done by rinsing in water.

In some embodiments, the methods described herein also eliminate theneed for chemical or mechanical polishing after the deposition of thegel material.

In some embodiments, the methods further include covalently attaching anucleic acid (e.g., oligonucleotide) to the gel material.

Lift-Off Gel Patterning

Some further embodiments described herein are related to methods ofpreparing a patterned surface by a Lift-Off gel patterning method. Thismethod can include using solid support having a continuous hydrophobiccoating layer and disposing a photoresist layer on the hydrophobiccoating layer. The photoresist can then be patterned by photolithographyusing a photomask comprising a plurality of micro-scale or nano-scalepatterns such that the micro-scale or nano-scale patterns aretransferred to the surface to form micro-scale or nano-scale contours onthe surface separated by hydrophobic interstitial regions. In someembodiments, the micro-scale or nano-scale contours are formed byetching off portions of the hydrophobic coating layer. A layer of silanecan then be applied to the surface to cover at least a portion of thecontours and a portion of the hydrophobic interstitial regions and a gelmaterial can be covalently attached to the layer of silane. Thesemethods are also known as lift-off methods.

In the lift-off methods described, rather than removing the photoresistright after pattern transfer as described in the direct patterningprocess, the methods can directly apply a layer of silane to the surfaceto cover at least a portion of the contours and a portion of thehydrophobic interstitial regions in the presence of the photoresist.Then, a gel material may be covalently attached to the layer of silane.In some embodiments, the methods further include curing the gelmaterial. The curing process may be done at various conditions,depending on the type of gel material used, as understood by one ofordinary skill in the art. In some embodiments, the curing is done byincubating the gel material deposited on the silane layer in an oven.

After the gel material is immobilized on the silane layer, the remainingphotoresist in the interstitial regions can then be removed or “liftedoff” thereby exposing the underlying hydrophobic coating layer. Theremaining photoresist may be removed by various reagents, depending onthe type of photoresist used. For example, photoresist LOR may beremoved by MICROCHEM Remover PG. In some embodiments, the remainingphotoresist is removed by acetone, for example, sonication in acetonesolution. The second adhesion promoting layer may be removed with thephotoresist at the same time. Alternatively, the second adhesionpromoting layer is removed subsequent to the photoresist using adifferent removal reagent. In one embodiment, the photoresist and thesecond adhesion promoting layer are both removed by acetone.

In some embodiments, the methods further include removing excess gelmaterial that is not immobilized to the silane layer.

In some embodiments, the methods further include covalently attaching anucleic acid (e.g. an oligonucleotide) to the gel material.

In methods or compositions set forth herein (e.g., surface preparation,direct patterning, and lift-off methods), the silane used herein maycomprise functional groups to forming covalent bonding with the gelmaterials. Non-limiting examples of the functional groups in the silaneinclude vinyl, acryloyl, alkenyl, cycloalkenyl, heterocycloalkenyl,alkynyl, cycloalkynyl, heterocycloalkynyl, nitrene, aldehyde,hydrazinyl, glycidyl ether, epoxy, carbene, isocyanate or maleimide, oroptionally substituted variants or combinations thereof. For example,the silane used herein may comprise an amino group (such as APTES orAPTMS). In some preferred embodiment, the silane used herein comprisesnorbornene derivatized silane. In one embodiment, the silane comprises[(5-bicyclo[2.2.1]hept-2-enyl)ethyl]trimethoxysilane. The silane may bedeposited on the surface of the solid support via chemical vapordeposition.

In methods or compositions set forth herein (e.g., surface preparation,direct patterning, and lift-off methods), the gel material that may beused includes, but is not limited to hydrogels or polymers. Usefulhydrogels include, but are not limited to, silane-free acrylamide (SFA)polymer, poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide)(PAZAM), polyacrylamide polymers formed from acrylamide and an acrylicacid or an acrylic acid containing a vinyl group as described, forexample, in WO 00/31148 (incorporated herein by reference);polyacrylamide polymers formed from monomers that form [2+2]photo-cycloaddition reactions, for example, as described in WO 01/01143or WO 03/014392 (each of which is incorporated herein by reference); orpolyacrylamide copolymers described in U.S. Pat. No. 6,465,178, WO01/62982 or WO 00/53812 (each of which is incorporated herein byreference). Chemically-treated variants of these gel materials are alsouseful, such as a hydrogel having reactive sites that is capable ofreacting with oligonucleotides having corresponding reactive groups (forexample, PAZAM is capable of reacting with a 5′- or 3′-alkynyl modifiedoligonucleotides). Other useful gels are those that are formed by atemperature dependent change in state from liquid to gelatinous.Examples include, but are not limited to agar, agarose, or gelatin. Insome embodiments, the gel material is covalently attached to the silanelayer.

In some embodiments, a gel material that is used will include reactivesites. The term “reactive site” as used herein means a site on the geldescribed herein that can be used to attach one or more molecules to thegel material, and/or to attach the gel material to the surface, by wayof a chemical reaction or molecular interaction. Non-limiting examplesof reactive sites include azido, optionally substituted amino,Boc-protected amino, hydroxy, thiol, alkynyl, alkenyl, halo, epoxy,tetrazinyl, or aldehyde. In some embodiments, the gel material comprisesa polymer with azido functional groups as reactive sites. In particularembodiments, the gel material comprises PAZAM. PAZAM is capable ofreacting with norbornene-derivatized silane to form covalent bonding viacatalyst free strain-promoted cycloaddition. In some embodiments, thereactive sites of the gel material are also capable of forming covalentbonding with functionalized oligo nucleotides for the purpose of primergrafting. In some alternative embodiments, the gel material ispre-grafted with primers before reacting with the silane layer.

In some other embodiments, a gel-forming (e.g., polymerizable) materialmay be provided on the surface in a liquid state and subsequentlyconverted to a gel. Examples of polymerizable materials include, withoutlimitation, acrylamide, methacrylamide, hydroxyethyl methacrylate,N-vinyl pyrolidinone, or derivatives thereof. Such materials are usefulfor preparing hydrogels. In some embodiments, the polymerizable materialcan include two or more different species of compound that form aco-polymer. For example, two or more different species of acrylamide,methacrylamide, hydroxyethyl methacrylate, N-vinyl pyrolidinone, orderivatives thereof can function as co-monomers that polymerize to forma copolymer hydrogel.

Patterned Surfaces and Solid Supports

Described herein is a patterned surface with gel-coated contourscomprising a solid support comprising:

-   -   a surface, the surface comprising a continuous hydrophobic        coating layer;    -   a photoresist layer on the hydrophobic coating layer of the        solid support, wherein the photoresist layer comprises        micro-scale or nano-scale contours; and    -   a layer of gel material within the micro-scale or nano-scale        contours, wherein the gel material is capable of covalently        bonding to oligonucleotides.

In some embodiments, the contours are separated by hydrophobicinterstitial regions, and further comprise a layer of binding material(such as a layer of silane) covering at least a portion of the contoursand a portion of the hydrophobic interstitial regions. In someembodiments, at least a portion of the micro-scale or nano-scalecontours are free of the hydrophobic coating. In some embodiments, thecontours comprise wells. In some embodiments, the surface comprises ahydrophobic coating layer, a photoresist layer on the hydrophobiccoating layer of the solid support, wherein the photoresist layercomprises micro-scale or nano-scale contours; a layer of bindingmaterial or silane covering at least a portion of the contours and atleast a portion of the hydrophobic interstitial regions.

In some embodiments, the hydrophobic coating layer comprises afluorinated polymer, a perfluorinated polymer, or a silicon polymer, ora mixture thereof. In some embodiments, the hydrophobic coating layercomprises an amorphous fluoropolymer, CYTOP-M, CYTOP-S, CYTOP-A, apolytetrafluoroethylene, Teflon, parylen, a fluorinated hydrocarbon, afluoroacrylic copolymer, Cytonix Fluoropel, a fluorosilane, aplasma-deposited fluorocarbon, a silicon polymer, apolydimethylsiloxane, or a siloxane, or a mixture thereof. In otherembodiments, the hydrophobic coating layer comprises a perfluorinatedpolymer. In other embodiments, the hydrophobic coating layer comprisesCYTOP-M, CYTOP-S, or CYTOP-A, or a mixture thereof. In otherembodiments, the hydrophobic coating layer comprises CYTOP-S. In otherembodiments, the hydrophobic coating layer comprises CYTOP-M. In otherembodiments, the hydrophobic coating layer comprises CYTOP-S andCYTOP-A.

In some embodiments, the hydrophobic coating layer is in direct contactwith the surface. In other embodiments, the hydrophobic coating layer isin contact with the surface via a first adhesion promoting layer. Insome embodiments, the first adhesion promoting layer comprises CYTOP-A,APTMS, or APTES, or a combination thereof.

In some embodiments, the photoresist is in direct contact with thehydrophobic coating layer of the solid support. In other embodiments,the photoresist is in contact with the hydrophobic coating layer of thesolid support via a second adhesion promoting layer. In someembodiments, the second adhesion promoting layer comprises a fluorinatedsurfactant. In some embodiments, the fluorinated surfactant is SurflonS-651, Novec FC-4430, Novec FC-4432, Novec FC-4434, Novec FC-5210, ZonylFSN-100, Zonyl FS-300, Zonyl FS-500, Capstone FS-10, Capstone FS-30,Capstone FS-60, Capstone FS-61, Capstone FS-63, Capstone FS-64, orCapstone FS-65, or a combination thereof.

In some embodiments, the photoresist is a Shipley S1800™ seriesphotoresist. In some embodiments, the photoresist is selected fromShipley S1818 (MICROPOSIT™ S1818™) and Shipley S1805 (MICROPOSIT™S1805™).

In some embodiments, the gel material comprises PAZAM. In someembodiments, the gel material comprises PAZAM attached to nucleic acids.

In some embodiments, the surface further comprises (a) a bindingmaterial layer or (b) a silane layer, wherein the binding material layeror silane layer optionally comprises a norbornene derivatized silane,and wherein the binding material layer or silane layer covers at least aportion of the contours and a portion of the hydrophobic interstitialregions.

Also described herein are methods of preparing such patterned surfaces.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art. The use of the term “including” as well as other forms, suchas “include,” “includes,” and “included,” is not limiting. The use ofthe term “having” as well as other forms, such as “have,” “has,” and“had,” is not limiting. As used in this specification, whether in atransitional phrase or in the body of the claim, the terms “comprise(s)”and “comprising” are to be interpreted as having an open-ended meaning.That is, the above terms are to be interpreted synonymously with thephrases “having at least” or “including at least.” For example, whenused in the context of a process, the term “comprising” means that theprocess includes at least the recited steps, but may include additionalsteps. When used in the context of a compound, composition, or device,the term “comprising” means that the compound, composition, or deviceincludes at least the recited features or components, but may alsoinclude additional features or components.

As used herein, common abbreviations are defined as follows:

APTS Aminopropyl silane

APTES (3-Aminopropyl)triethoxysilane

APTMS (3-Aminopropyl)trimethoxysilane

aq. Aqueous

Azapa N-(5-azidoacetamidylpentyl) acrylamide

° C. Temperature in degrees Centigrade

CA Contact angle

CVD Chemical vapor deposition

dATP Deoxyadenosine triphosphate

dCTP Deoxycytidine triphosphate

dGTP Deoxyguanosine triphosphate

dTTP Deoxythymidine triphosphate

g Gram(s)

h or hr Hour(s)

IPA Isopropyl Alcohol

m or min Minute(s)

mL Milliliter(s)

PAZAM Poly(N-(5-azidoacetamidylpentyl) acrylamide-co-acrylamide) of anyacrylamide to Azapa ratio

rt Room temperature

SFA Silane Free Acrylamide as defined in U.S. Pat. Pub. No. 2011/0059865

SBS Sequencing-by-synthesis

SHP Semi-hydrophobic

ssDNA Single stranded DNA

As used herein, the term “attached” refers to the state of two thingsbeing joined, fastened, adhered, connected or bound to each other. Forexample, an analyte, such as a nucleic acid, can be attached to amaterial, such as a gel or solid support, by a covalent or non-covalentbond. A covalent bond is characterized by the sharing of pairs ofelectrons between atoms. A non-covalent bond is a chemical bond thatdoes not involve the sharing of pairs of electrons and can include, forexample, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilicinteractions and hydrophobic interactions.

As used herein, the term “array” refers to a population of differentprobes (e.g., probe molecules) that are attached to one or moresubstrates such that the different probes can be differentiated fromeach other according to relative location. An array can includedifferent probes that are each located at a different addressablelocation on a substrate. Alternatively or additionally, an array caninclude separate substrates each bearing a different probe, wherein thedifferent probes can be identified according to the locations of thesubstrates on a surface to which the substrates are attached oraccording to the locations of the substrates in a liquid. Exemplaryarrays in which separate substrates are located on a surface include,without limitation, those including beads in wells as described, forexample, in U.S. Pat. No. 6,355,431 B1, US 2002/0102578 and PCTPublication No. WO 00/63437. Exemplary formats that can be used in theinvention to distinguish beads in a liquid array, for example, using amicrofluidic device, such as a fluorescent activated cell sorter (FACS),are described, for example, in U.S. Pat. No. 6,524,793. Further examplesof arrays that can be used in the invention include, without limitation,those described in U.S. Pat. Nos. 5,429,807; 5,436,327; 5,561,071;5,583,211; 5,658,734; 5,837,858; 5,874,219; 5,919,523; 6,136,269;6,287,768; 6,287,776; 6,288,220; 6,297,006; 6,291,193; 6,346,413;6,416,949; 6,482,591; 6,514,751 and 6,610,482; WO 93/17126; WO 95/11995;WO 95/35505; EP 742 287; and EP 799 897.

As used herein, the term “covalently attached” or “covalently bonded”refers to the forming of a chemical bonding that is characterized by thesharing of pairs of electrons between atoms. For example, a covalentlyattached hydrogel refers to a hydrogel that forms chemical bonds with afunctionalized surface of a substrate, as compared to attachment to thesurface via other means, for example, adhesion or electrostaticinteraction. It will be appreciated that polymers that are attachedcovalently to a surface can also be bonded via means in addition tocovalent attachment.

As used herein, the term “coat,” when used as a verb, is intended tomean providing a layer or covering on a surface. At least a portion ofthe surface can be provided with a layer or cover. In some cases theentire surface can be provided with a layer or cover. In alternativecases only a portion of the surface will be provided with a layer orcovering. The term “coat,” when used to describe the relationshipbetween a surface and a material, is intended to mean that the materialis present as a layer or cover on the surface. The material can seal thesurface, for example, preventing contact of liquid or gas with thesurface. However, the material need not form a seal. For example, thematerial can be porous to liquid, gas, or one or more components carriedin a liquid or gas. Exemplary materials that can coat a surface include,but are not limited to, a gel, polymer, organic polymer, liquid, metal,a second surface, plastic, silica, or gas.

As used herein the term “analyte” is intended to include any of avariety of analytes that are to be detected, characterized, modified,synthesized, or the like. Exemplary analytes include, but are notlimited to, nucleic acids (e.g., DNA, RNA or analogs thereof), proteins,polysaccharides, cells, nuclei, cellular organelles, antibodies,epitopes, receptors, ligands, enzymes (e g kinases, phosphatases orpolymerases), peptides, small molecule drug candidates, or the like. Anarray can include multiple different species from a library of analytes.For example, the species can be different antibodies from an antibodylibrary, nucleic acids having different sequences from a library ofnucleic acids, proteins having different structure and/or function froma library of proteins, drug candidates from a combinatorial library ofsmall molecules, etc.

As used herein the term “contour” is intended to mean a localizedvariation in the shape of a surface. Exemplary contours include, but arenot limited to, wells, pits, channels, posts, pillars, and ridges.Contours can occur as any of a variety of depressions in a surface orprojections from a surface. All or part of a contour can serve as afeature in an array. For example, a part of a contour that occurs in aparticular plane of a solid support can serve as a feature in thatparticular plane. In some embodiments, contours are provided in aregular or repeating pattern on a surface.

Where a material is “within” a contour, it is located in the space ofthe contour. For example, for a well, the material is inside the well,and for a pillar or post, the material covers the contour that extendsabove the plane of the surface.

In some embodiments, where a second layer is said to “cover” a firstlayer, the second layer is in the form of a thin film on top of thefirst layer.

As used herein, the term “different”, when used in reference to nucleicacids, means that the nucleic acids have nucleotide sequences that arenot the same as each other. Two or more nucleic acids can havenucleotide sequences that are different along their entire length.Alternatively, two or more nucleic acids can have nucleotide sequencesthat are different along a substantial portion of their length. Forexample, two or more nucleic acids can have target nucleotide sequenceportions that are different for the two or more molecules while alsohaving a universal sequence portion that is the same on the two or moremolecules. The term can be similarly applied to proteins which aredistinguishable as different from each other based on amino acidsequence differences.

As used herein, the term “each,” when used in reference to a collectionof items, is intended to identify an individual item in the collectionbut does not necessarily refer to every item in the collection.Exceptions can occur if explicit disclosure or context clearly dictatesotherwise.

As used herein, the term “feature” means a location in an array that isconfigured to attach a particular analyte. For example, a feature can beall or part of a contour on a surface. A feature can contain only asingle analyte or it can contain a population of several analytes,optionally the several analytes can be the same species. In someembodiments, features are present on a solid support prior to attachingan analyte. In other embodiments the feature is created by attachment ofan analyte to the solid support.

As used herein, the term “flow cell” is intended to mean a vessel havinga chamber where a reaction can be carried out, an inlet for deliveringreagents to the chamber and an outlet for removing reagents from thechamber. In some embodiments, the chamber is configured for detection ofthe reaction that occurs in the chamber (e.g. on a surface that is influid contact with the chamber). For example, the chamber can includeone or more transparent surfaces allowing optical detection of arrays,optically labeled molecules, or the like in the chamber. Exemplary flowcells include, but are not limited to those used in a nucleic acidsequencing apparatus such as flow cells for the Genome Analyzer®,MiSeq®, NextSeq® or HiSeq® platforms commercialized by Illumina, Inc.(San Diego, Calif.); or for the SOLiD™ or Ion Torrent™ sequencingplatform commercialized by Life Technologies (Carlsbad, Calif.).Exemplary flow cells and methods for their manufacture and use are alsodescribed, for example, in WO 2014/142841 A1; U.S. Pat. App. Pub. No.2010/0111768 A1 and U.S. Pat. No. 8,951,781, each of which isincorporated herein by reference.

As used herein, the term “gel material” is intended to mean a semi-rigidmaterial that is permeable to liquids and gases. Typically, a gelmaterial can swell when liquid is taken up and can contract when liquidis removed, e.g., by drying. Exemplary gels include, but are not limitedto, those having a colloidal structure, such as agarose; polymer meshstructure, such as gelatin; or cross-linked polymer structure, such aspolyacrylamide, silane free acrylamide (see, for example, US Pat. App.Pub. No. 2011/0059865 A1), PAZAM (see, for example, U.S. Pat. No.9,012,022, which is incorporated herein by reference), and polymersdescribed in U.S. Patent Pub. No. 2015/0005447, and U.S. applicationSer. No. 14/927,252, all of which are incorporated by reference in theirentireties. Particularly useful gel material will conform to the shapeof a well or other contours where it resides. Some useful gel materialscan both (a) conform to the shape of the well or other contours where itresides and (b) have a volume that does not substantially exceed thevolume of the well or contours where it resides. In some particularembodiments, the gel material is a polymeric hydrogel.

As used herein, the term “interstitial region” refers to an area in asubstrate or on a surface that separates other areas of the substrate orsurface. For example, an interstitial region can separate one contour orfeature from another contour or feature on the surface. The two regionsthat are separated from each other can be discrete, lacking contact witheach other. In many embodiments the interstitial region is continuouswhereas the contours or features are discrete, for example, as is thecase for an array of wells in an otherwise continuous surface. Theseparation provided by an interstitial region can be partial or fullseparation. Interstitial regions will typically have a surface materialthat differs from the surface material of the contours or features onthe surface. For example, contours of an array can have an amount orconcentration of gel material or analytes that exceeds the amount orconcentration present at the interstitial regions. In some embodimentsthe gel material or analytes may not be present at the interstitialregions.

As used herein, the terms “nucleic acid” and “nucleotide” are intendedto be consistent with their use in the art and to include naturallyoccurring species or functional analogs thereof. Particularly usefulfunctional analogs of nucleic acids are capable of hybridizing to anucleic acid in a sequence specific fashion or capable of being used asa template for replication of a particular nucleotide sequence.Naturally occurring nucleic acids generally have a backbone containingphosphodiester bonds. An analog structure can have an alternate backbonelinkage including any of a variety of those known in the art. Naturallyoccurring nucleic acids generally have a deoxyribose sugar (e.g. foundin deoxyribonucleic acid (DNA)) or a ribose sugar (e.g., found inribonucleic acid (RNA)). A nucleic acid can contain nucleotides havingany of a variety of analogs of these sugar moieties that are known inthe art. A nucleic acid can include native or non-native nucleotides. Inthis regard, a native deoxyribonucleic acid can have one or more basesselected from the group consisting of adenine, thymine, cytosine orguanine and a ribonucleic acid can have one or more bases selected fromthe group consisting of uracil, adenine, cytosine or guanine. Usefulnon-native bases that can be included in a nucleic acid or nucleotideare known in the art. The terms “probe” or “target,” when used inreference to a nucleic acid, are intended as semantic identifiers forthe nucleic acid in the context of a method or composition set forthherein and does not necessarily limit the structure or function of thenucleic acid beyond what is otherwise explicitly indicated. The terms“probe” and “target” can be similarly applied to other analytes such asproteins, small molecules, cells, or the like.

As used herein, the term “fluorinated” refers to a molecule containingat least one fluorine atom. As used herein, the term “perfluorinated”refers to a molecule containing two or more fluorine atoms. In someembodiments, perfluorinated molecules are hydrocarbon-containingmolecules in which the hydrogen atoms on sp3-hybridized carbons arereplaced with fluorine atoms. For example, certain perfluorinatedpolymers described herein contain a perfluoroalkyl group or aperfluoroalkylene moiety.

As used herein, the term “photoresist” and derivatives thereof refers toa light-sensitive material used in processes such as photolithography,photoetching, or photoengraving to form a patterned coating on asurface. Photoresist materials change solubility with respect to adeveloper solution when exposed to certain wavelengths of light.Photoresist layers may be composed of positive (exposed region becomessoluble) or negative (exposed region becomes insoluble) photoresistmaterial.

As used herein, the term “pitch,” when used in reference to contours orfeatures on a surface, is intended to refer to the center-to-centerspacing for adjacent features. A pattern of features can becharacterized in terms of average pitch. The pattern can be ordered suchthat the coefficient of variation around the average pitch is small orthe pattern can be random in which case the coefficient of variation canbe relatively large. In either case, the average pitch can be, forexample, at least about 10 nm, 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 1μm, 5 μm, 10 μm, 100 μm or more, or a range defined by any of the twopreceding values (e.g., 10 to 100 nm, 10 to 200 nm, 200 to 400 nm, 300to 500 nm). Alternatively or additionally, the average pitch can be, forexample, at most about 100 μm, 10 μm, 5 μm, 1 μm, 0.5 μm, 0.1 μm orless, or a range defined by any of the two preceding values. Of course,the average pitch for a particular pattern of features can be betweenone of the lower values and one of the upper values selected from theranges above.

As used herein, the term “repeating pattern,” when used in reference tofeatures, is intended to mean that the relative locations of a subset offeatures or contours in one region of the object is the same as therelative locations of a subset of features or contours in at least oneother region of the object. Generally, the repeat occurs in the x and ydimensions. The one region is typically adjacent to that other region inthe pattern. The relative locations for features in one region of arepeating pattern are generally predictable from the relative locationsof contours in another region of the repeating pattern. The subset usedfor the measure will generally include at least 2 features but caninclude at least, 3, 4, 5, 6, 10 or more features. Alternatively oradditionally, the subset used for the measure can include no more than2, 3, 4, 5, 6, or 10 features. Exemplary repeating patterns includesquare lattices, rectangular lattices, rhombic lattices, hexagonallattices and oblique lattices. A repeating pattern can include multiplerepetitions of a sub-pattern.

As used herein, the term “segregate,” when used in reference to a gelmaterial on two contours (or at two separate features), means toseparate or isolate the gel material on one of the contours (or at oneof the features) from the gel material on the other contour (or at theother feature). Thus, the gel material on the first contour (or at thefirst feature) is not in direct contact with the gel material in theother well (or at the other feature). In some embodiments, the term“segregate” is used in reference to a gel material in two wells, andmeans to separate or isolate the gel material in one of the well fromthe gel material in the other well. In some embodiments, the gelmaterial in the two wells (or at the two features) is in indirectcontact, for example, via a solution that contacts the two wells (orfeatures). Alternatively, the gel material in the two wells (or at thetwo features) is not even in indirect contact. An interstitial region ona surface can segregate the gel material in two wells (or at twofeatures) by being devoid of the gel material. In particularembodiments, a gel material can be discontinuous on a surface, beingpresent at features, such as wells, but not present at interstitialregions between the features.

As used herein, the term “surface” is intended to mean an external partor external layer of a solid support or gel material. The surface can bein contact with another material such as a gas, liquid, gel, polymer,organic polymer, second surface of a similar or different material,metal, or coat. The surface, or regions thereof, can be substantiallyflat or planar. The surface can have surface contours such as wells,pits, channels, ridges, raised regions, pegs, posts or the like.

As used herein, the term “solid support” refers to a rigid substratethat is insoluble in aqueous liquid. The substrate can be non-porous orporous. The substrate can optionally be capable of taking up a liquid(e.g., due to porosity) but will typically be sufficiently rigid thatthe substrate does not swell substantially when taking up the liquid anddoes not contract substantially when the liquid is removed by drying. Anonporous solid support is generally impermeable to liquids or gases.Exemplary solid supports include, but are not limited to, glass andmodified or functionalized glass, plastics (e.g., acrylics, polystyreneand copolymers of styrene and other materials, polypropylene,polyethylene, polybutylene, polyurethanes, Teflon™, cyclic olefins,polyimides, etc.), nylon, ceramics, resins, Zeonor, silica orsilica-based materials including silicon and modified silicon, carbon,metals, inorganic glasses, optical fiber bundles, and polymers.Particularly useful solid supports for some embodiments are componentsof a flow cell or located within a flow cell apparatus.

As used herein, the term “well” refers to a discrete contour in a solidsupport having a surface opening that is completely surrounded byinterstitial region(s) of the surface. Wells can have any of a varietyof shapes at their opening in a surface including but not limited toround, elliptical, square, polygonal, star shaped (with any number ofvertices), etc. The cross section of a well taken orthogonally with thesurface can be curved, square, polygonal, hyperbolic, conical, angular,etc. In some embodiments, the well is a microwell or a nanowell.

The embodiments set forth herein and recited in the claims can beunderstood in view of the above definitions.

FIG. 1 shows three types of fluoropolymers that may be used to createthe hydrophobic interstitial regions of the patterned surface of asubstrate. CYTOP-A, CYTOP-M, and CYTOP-S are commercially availableamorphous perfluorinated polymers, each having the following backbonestructure:

and different functional groups at both ends of the polymer chain.CYTOP-A has —C(O)OH end functional group. CYTOP-M has —C(O)NH—Si(OR)_(n)functional group. CYTOP-S has —CF₃ functional groups. FIG. 1 also showsthe type of possible interactions between each type of CYTOP polymer andthe surface. It indicates that CYTOP-S has no chemical interaction withmetal surface, silane finished surface, or Si/SiN surface due to theinertness of the —CF₃ functional group.

FIG. 2A is a diagrammatic cross section partial view of a patternedsurface of a glass substrate 10. As shown, the glass substrate 10 has atop surface 15 and bottom surface 20. The top surface 15 has a pluralityof wells 25 a-25 d formed in the top surface 15. Each of the wells 25a-25 d has a polymer material or gel 30 a-30 d lining the interior wallsof the wells. A series of hydrophobic interstitial regions 35 a-35 e areshown on the top surface 15 located between each of the wells 25 a-25 d.As shown, the polymer material or gel 30 a-30 d is deposited on thebottom of the wells 25 a-25 d and DNA clusters 40 a-40 d are covalentlyattached to the gel material 30 a-30 d.

FIG. 2B is a more detailed diagrammatic example of the surfacesdescribed herein. As illustrated, the surface includes three layers ofmaterials on top of the glass slide. The bottom CYTOP layer is in directcontact with the bottom glass surface. Deposited on top of the CYTOPmaterial is a norbornene derivatized silane layer. On top of thenorbornene layer is a top layer comprising PAZAM. A set of grafted P5/P7oligonucleotide primers with the fluorophore TET™ attached are bound tothe PAZAM layer of material. Sequences of P5 and P7 primers are setforth in U.S. Pat. No. 8,969,258, which is incorporated herein byreference.

FIG. 2C are fluorescence images of three grafted glass surfaces. Achemical inertness test was conducted on CYTOP-S and CYTOP-M. The leftsurface was used as a control and was not coated with any CYTOPpolymers. The middle surface was coated with CYTOP-M and the rightsurface was coated with CYTOP-S. Then, each surface was treated with anorbornene derivatized silane, followed by PAZAM coupling and graftingof P5 and P7 oligonucleotides to the PAZAM. The presence or absence ofPAZAM attached oligonucleotides was evaluated by hybridization offluorescently labelled TET oligonucleotides (complementary to P5 and P7)and detection on a Typhoon fluorescence imager. The lack of TET oligointensity on CYTOP-S treated surface indicates thatoligonucleotide-grafted PAZAM was not immobilized on the CYTOP-S treatedsurface.

FIG. 3 illustrates the contact angles of the CYTOP-M and CYTOP-S treatedglass surface compared to the control surface before and after chemicaltreatment. CYTOP treated glass surface showed a contact angle of about120 degrees for CYTOP-M and about 123 degrees for CYTOP-S. After thedeposition of the norbornene silane layer, the contact angle decreasedto 116 degrees for CYTOP-M and 120 degrees for CYTOP-S. Following PAZAMcoupling and oligo grafting, the contact angle decreased substantiallydown to 51 degrees for the CYTOP-M surface, indicating that DNA clustersand the hydrophilic PAZAM polymer bound to the surface and the surfacewas rendered hydrophilic. In contrast, CYTOP-S surface retained itshydrophobicity with only a slight decrease in contact angle. Due to itschemical inertness and hydrophobic characteristics, CYTOP-S isidentified to be a good candidate for surface patterning (e.g. to forminterstitial regions between analyte-bearing features).

A typical workflow is illustrated in FIG. 4 for preparing a patternedsurface using CYTOP-S as hydrophobic coating. First, the surface wastreated with CYTOP-S. Because CYTOP-S lacks any reactive functionalgroups to couple to the glass or silicon surface, an adhesion promotinglayer may be used to facilitate the coating of CYTOP-S to the surface.In this example, a thin layer of CYTOP-A was first coated to the glasssurface. Then, CYTOP-S was coated evenly on the surface. Aftersubsequent curing, the perfluorinated polymers in the two CYTOP layersentangle further to form stronger adhesion. Non-limiting examples of amaterial that may be used as an adhesion promoting layer for glass orsilicon surface also include an amino-based silane coupling agent suchas APTMS, APTES, etc.

In this standard workflow, the CYTOP-S surface was treated with oxygenplasma in order to deposit standard photoresist for photolithography.The plasma treatment makes the CYTOP-S more hydrophilic so that thephotoresist can be coated on top of it. With this surface modification,standard photoresists tend to dewet from the surface. During theprocess, the CYTOP-S surface lost its hydrophobicity and chemicalinertness after the oxygen plasma treatment. While the hydrophobicity ofCYTOP-S surface was recovered via a high temperature reflow step at 180°C. using a CYTOP-S solution, the chemical inertness of the surface wasnot recovered and non-specific binding of PAZAM to the interstitialCYTOP-S surface was observed.

Reflow Process

In any embodiments of the methods described herein, the methods mayfurther comprise a reflow process to recover damage to the surfaceduring patterning. For example, if the CYTOP-S surface has been damaged(such as loss of inertness or of hydrophobic properties) during thepatterning process, CYTOP-S surface properties may be restored by areflow process using a CYTOP-S containing solvent. The solvent reflowmay be conducted either as a liquid phase process as exemplified in FIG.9A or as a vapor phase process as exemplified in FIG. 9B. Non-limitingexamples of a liquid phase reflow include depositing the CYTOP-S solventon surface or spin coating on the surface directly at a high temperature(for example, 180° C.), then curing at a lower temperature (for example,50° C.). In the vapor phase reflow, the substrate is placed in avacuum-sealed desiccator with some amount of CYTOP-S containing solventin it, such as 2 mL of a perfluorinated fluorocarbon solvent such asCT-SOLV100E. Solvents that may be used in the reflow process include butare not limited to CT-SOLV180, or CT-SOLV100E. Other solvents of choiceinclude Fluorinert™ FC-40, Fluorinert™ FC-770, each of which is capableof dissolving the perfluorinated polymers.

In another embodiment, the patterned CYTOP-S surface may be subject tothe reflow process after hydrogel coating to restore surface propertydamage. The reflow process has no impact on the hydrogel quality forsequencing. FIG. 10A is an optical microscopy image of PAZAM coatedCYTOP-S nanowells (700 nm to 1.1 μm) after O₂ plasma treatment. FIG. 10Bis an optical microscopy image of the PAZAM coated CYTOP-S nanowells inFIG. 10A after liquid phase solvent reflow. FIG. 10C is a fluorescenceimage of the PAZAM coated CYTOP-S nanowells of FIG. 10B after thewetting/dewetting of an aqueous droplet with fluorescent die, indicatingthat PAZAM is still accessible.

Solid Support

Solid supports that are useful in an apparatus or method of the presentdisclosure can be a generally flat surface (e.g., a chip or slide) orcan have a curved surface (e.g. a cylinder or drum). It can also be two-or three-dimensional. Useful materials include glass, quartz, plastic(such as polystyrene (low cross-linked and high cross-linkedpolystyrene), polycarbonate, polypropylene or poly(methylmethacrylate)),acrylic copolymer, polyamide, silicon, metal (e.g.,alkanethiolate-derivatized gold), cellulose, nylon, latex, dextran, gelmatrix (e.g., silica gel), polyacrolein, or composites. In someembodiments, the solid support comprises glass.

Features

The features of an array can have any of a variety of shapes. In someembodiments, the term “feature” also refers to “contours” on a patternedsurface when all of a contour serves as a feature in an array. Forexample, when observed in a two dimensional plane, such as on thesurface of an array, the features can appear rounded, circular, oval,rectangular, square, symmetric, asymmetric, triangular, polygonal, orthe like. The features can be arranged in a regular repeating patternincluding, for example, a square lattice, rectangular lattice, rhombiclattice, hexagonal lattice or oblique lattice. A pattern can be selectedto achieve a desired level of packing. For example, round features areoptimally packed in a hexagonal arrangement. Of course other packingarrangements can also be used for round features and vice versa.

The size of a feature on an array (or other object used in a method orsystem herein) can be selected to suit a particular application. Forexample, in some embodiments a feature of an array can have a size thataccommodates only a single nucleic acid molecule. A surface having aplurality of features in this size range is useful for constructing anarray of molecules for detection at single molecule resolution. Featuresin this size range are also useful for use in arrays having featuresthat each contain a colony of nucleic acid molecules. Thus, the featuresof an array can each have an area that is no larger than about 1 mm², nolarger than about 500 μm², no larger than about 100 μm², no larger thanabout 10 μm², no larger than about 1 μm², no larger than about 500 nm²,or no larger than about 100 nm², no larger than about 10 nm², no largerthan about 5 nm², or no larger than about 1 nm². Alternatively oradditionally, the features of an array will be no smaller than about 1mm², no smaller than about 500 μm², no smaller than about 100 μm², nosmaller than about 10 μm², no smaller than about 1 μm², no smaller thanabout 500 nm², no smaller than about 100 nm², no smaller than about 10nm², no smaller than about 5 nm², or no smaller than about 1 nm².Indeed, a feature can have a size that is in a range between an upperand lower limit selected from those exemplified above. Although severalsize ranges for features of a surface have been exemplified with respectto nucleic acids and on the scale of nucleic acids, it will beunderstood that features in these size ranges can be used forapplications that do not include nucleic acids. It will be furtherunderstood that the size of the features need not necessarily beconfined to a scale used for nucleic acid applications.

An array can also be characterized with regard to pitch. For example,the size of the features and/or pitch of the features can vary such thatarrays can have a desired density. For example, the average featurepitch can be at most 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, 0.5 μm, 0.4 μm,0.3 μm, 0.2 μm, 0.1 μm or less. Alternatively or additionally, theaverage feature pitch can be at least 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm,0.5 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm or more. Similarly, the maximumfeature pitch can be at most 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, 0.5 μm0.4 μm, 0.3 μm, 0.2 μm, 0.1 μm or less; and/or the minimum feature pitchcan be at least 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 1 μm, 5 μm, 10μm, 50 μm, 100 μm or more. The above ranges can apply to the average,maximum or minimum pitch between features.

The density of features in an array can also be understood in terms ofthe number of features present per unit area. For example, the averagedensity of features for an array can be at least about 1×10³features/mm², 1×10⁴ features/mm², 1×10⁵ features/mm², 1×10⁶features/mm², 1×10⁷ features/mm², 1×10⁸ features/mm², or 1×10⁹features/mm² or higher. Alternatively or additionally the averagedensity of features for an array can be at most about 1×10⁹features/mm², 1×10⁸ features/mm², 1×10⁷ features/mm², 1×10⁶features/mm², 1×10⁵ features/mm², 1×10⁴ features/mm², or 1×10³features/mm² or less.

An array having a regular pattern of features can be ordered withrespect to the relative locations of the features but random withrespect to one or more other characteristic of each feature. Forexample, in the case of a nucleic acid array, the nucleic acid featurescan be ordered with respect to their relative locations but random withrespect to one's knowledge of the sequence for the nucleic acid speciespresent at any particular feature. As a more specific example, nucleicacid arrays formed by seeding a repeating pattern of features withtemplate nucleic acids and amplifying the template at each feature toform copies of the template at the feature (e.g., via clusteramplification or bridge amplification) will have a regular pattern ofnucleic acid features, as determined by the position of the contoursthat form the features, but will be random with regard to thedistribution of sequences of the nucleic acids across the array. Thus,detection of the presence of nucleic acid material generally on thearray can yield a repeating pattern of features, whereas sequencespecific detection can yield non-repeating distribution of signalsacross the array.

In some embodiments, the methods described herein form contours with asingle repeating pattern. In some other embodiments, the methodsdescribed herein form contours with multiple repeating patterns,providing arrays with at least a first repeating pattern of features anda second repeating pattern of features. In some such embodiments, thefirst and second patterns form an interleaved pattern along the exteriorsurface, wherein the features of the first repeating pattern occur at afirst elevation and the features of the second repeating pattern occurat a second elevation, and wherein the features include attachmentpoints for analytes, whereby the features of the first repeating patternare configured to attach analytes at a different elevation relative toanalytes attached to the features of the second repeating pattern.Examples of substrates having contours with multiple repeating patternsthat can be made or used in a method or composition set forth herein aredescribed in PCT Appln. No. PCT/US2017/024578, filed Mar. 28, 2017, andtitled “Multi-Plane Microarrays” which is hereby incorporated byreference in its entirety.

Analytical Applications

Some embodiments are directed to methods of detecting an analyte using asubstrate with a patterned surface prepared by the methods describedherein. In some embodiments, the analyte is selected from nucleic acids,polynucleotides, proteins, antibodies, epitopes to antibodies, enzymes,cells, nuclei, cellular organelles, or small molecule drugs. In oneembodiment, the analyte is a polynucleotide. In one embodiment, thedetecting includes determining a nucleotide sequence of thepolynucleotide.

Some embodiments described herein are related to methods of preparing anarray of polynucleotides, the methods include providing a solid supportcomprising a patterned surface, the surface comprising microscale and/ornanoscale contours coated with a gel material that is capable ofcovalently bonding to oligonucleotides, the surface is prepared by anyof the methods described herein; and covalently attaching a plurality offirst oligonucleotides and a plurality of second oligonucleotides to thegel material. In some embodiments, the methods further includecontacting the plurality of first oligonucleotides attached to thepolymer coating with templates to be amplified, each template comprisingat the 3′ end a sequence capable of hybridizing to the firstoligonucleotides and at the 5′ end a sequence the complement of which iscapable of hybridizing to the second oligonucleotides. In someembodiments, the methods further include amplifying the templates usingthe first oligonucleotides and the second oligonucleotides, therebygenerating a clustered array of polynucleotides.

Some embodiments that use nucleic acids can include a step of amplifyingthe nucleic acids on the substrate. Many different DNA amplificationtechniques can be used in conjunction with the substrates describedherein. Exemplary techniques that can be used include, but are notlimited to, polymerase chain reaction (PCR), rolling circleamplification (RCA), multiple displacement amplification (MDA), orrandom prime amplification (RPA). In particular embodiments, one or moreprimers used for amplification can be attached to a substrate (e.g. viaa gel or polymer coating). In PCR embodiments, one or both of theprimers used for amplification can be attached to the substrate. Formatsthat utilize two species of attached primer are often referred to asbridge amplification because double stranded amplicons form abridge-like structure between the two attached primers that flank thetemplate sequence that has been copied. Exemplary reagents andconditions that can be used for bridge amplification are described, forexample, in U.S. Pat. No. 5,641,658; U.S. Patent Publ. No. 2002/0055100;U.S. Pat. No. 7,115,400; U.S. Patent Publ. No. 2004/0096853; U.S. PatentPubl. No. 2004/0002090; U.S. Patent Publ. No. 2007/0128624; and U.S.Patent Publ. No. 2008/0009420, each of which is incorporated herein byreference.

PCR amplification can also be carried out with one amplification primerattached to a substrate and a second primer in solution. An exemplaryformat that uses a combination of one attached primer and soluble primeris emulsion PCR as described, for example, in Dressman et al., Proc.Natl. Acad. Sci. USA 100:8817-8822 (2003), WO 05/010145, or U.S. PatentPubl. Nos. 2005/0130173 or 2005/0064460, each of which is incorporatedherein by reference. Emulsion PCR is illustrative of the format and itwill be understood that for purposes of the methods set forth herein theuse of an emulsion is optional and indeed for several embodiments anemulsion is not used. Furthermore, primers need not be attached directlyto substrate or solid supports as set forth in the ePCR references andcan instead be attached to a gel or polymer coating as set forth herein.

RCA techniques can be modified for use in a method of the presentdisclosure. Exemplary components that can be used in an RCA reaction andprinciples by which RCA produces amplicons are described, for example,in Lizardi et al., Nat. Genet. 19:225-232 (1998) and US 2007/0099208 A1,each of which is incorporated herein by reference. Primers used for RCAcan be in solution or attached to a gel or polymer coating.

MDA techniques can be modified for use in a method of the presentdisclosure. Some basic principles and useful conditions for MDA aredescribed, for example, in Dean et al., Proc Natl. Acad. Sci. USA99:5261-66 (2002); Lage et al., Genome Research 13:294-307 (2003);Walker et al., Molecular Methods for Virus Detection, Academic Press,Inc., 1995; Walker et al., Nucl. Acids Res. 20:1691-96 (1992); U.S. Pat.Nos. 5,455,166; 5,130,238; and 6,214,587, each of which is incorporatedherein by reference. Primers used for MDA can be in solution or attachedto a gel or polymer coating.

In particular embodiments a combination of the above-exemplifiedamplification techniques can be used. For example, RCA and MDA can beused in a combination wherein RCA is used to generate a concatamericamplicon in solution (e.g. using solution-phase primers). The ampliconcan then be used as a template for MDA using primers that are attachedto a substrate (e.g. via a gel or polymer coating). In this example,amplicons produced after the combined RCA and MDA steps will be attachedto the substrate.

Substrates of the present disclosure that contain nucleic acid arrayscan be used for any of a variety of purposes. A particularly desirableuse for the nucleic acids is to serve as capture probes that hybridizeto target nucleic acids having complementary sequences. The targetnucleic acids once hybridized to the capture probes can be detected, forexample, via a label recruited to the capture probe. Methods fordetection of target nucleic acids via hybridization to capture probesare known in the art and include, for example, those described in U.S.Pat. Nos. 7,582,420; 6,890,741; 6,913,884 or 6,355,431 or U.S. Pat. Pub.Nos. 2005/0053980 A1; 2009/0186349 A1 or 2005/0181440 A1, each of whichis incorporated herein by reference. For example, a label can berecruited to a capture probe by virtue of hybridization of the captureprobe to a target probe that bears the label. In another example, alabel can be recruited to a capture probe by hybridizing a target probeto the capture probe such that the capture probe can be extended byligation to a labeled oligonucleotide (e.g., via ligase activity) or byaddition of a labeled nucleotide (e.g. via polymerase activity).

In some embodiments, a substrate described herein can be used fordetermining a nucleotide sequence of a polynucleotide. In suchembodiments, the method can comprise the steps of (a) contacting apolynucleotide polymerase with polynucleotide clusters attached to asurface of a substrate (e.g., via any one of the polymer or gel coatingsdescribed herein); (b) providing nucleotides to the surface of thesubstrate such that a detectable signal is generated when one or morenucleotides are utilized by the polynucleotide polymerase; (c) detectingsignals at one or more attached polynucleotide (or one or more clustersproduced from the attached polynucleotides); and (d) repeating steps (b)and (c), thereby determining a nucleotide sequence of asubstrate-attached polynucleotide.

Nucleic acid sequencing can be used to determine a nucleotide sequenceof a polynucleotide by various processes known in the art. In apreferred method, sequencing-by-synthesis (SBS) is utilized to determinea nucleotide sequence of a polynucleotide attached to a surface of asubstrate (e.g. via any one of the polymer coatings described herein).In such a process, one or more nucleotides are provided to a templatepolynucleotide that is associated with a polynucleotide polymerase. Thepolynucleotide polymerase incorporates the one or more nucleotides intoa newly synthesized nucleic acid strand that is complementary to thepolynucleotide template. The synthesis is initiated from anoligonucleotide primer that is complementary to a portion of thetemplate polynucleotide or to a portion of a universal or non-variablenucleic acid that is covalently bound at one end of the templatepolynucleotide. As nucleotides are incorporated against the templatepolynucleotide, a detectable signal is generated that allows for thedetermination of which nucleotide has been incorporated during each stepof the sequencing process. In this way, the sequence of a nucleic acidcomplementary to at least a portion of the template polynucleotide canbe generated, thereby permitting determination of the nucleotidesequence of at least a portion of the template polynucleotide.

Flow cells provide a convenient format for housing an array that isproduced by the methods of the present disclosure and that is subjectedto a sequencing-by-synthesis (SBS) or other detection technique thatinvolves repeated delivery of reagents in cycles. For example, toinitiate a first SBS cycle, one or more labeled nucleotides, DNApolymerase, etc., can be flowed into/through a flow cell that houses anucleic acid array made by methods set forth herein. Those sites of anarray where primer extension causes a labeled nucleotide to beincorporated can be detected. Optionally, the nucleotides can furtherinclude a reversible termination property that terminates further primerextension once a nucleotide has been added to a primer. For example, anucleotide analog having a reversible terminator moiety can be added toa primer such that subsequent extension cannot occur until a deblockingagent is delivered to remove the moiety. Thus, for embodiments that usereversible termination, a deblocking reagent can be delivered to theflow cell (before or after detection occurs). Washes can be carried outbetween the various delivery steps. The cycle can then be repeated ntimes to extend the primer by n nucleotides, thereby detecting asequence of length n. Exemplary SBS procedures, fluidic systems anddetection platforms that can be readily adapted for use with an arrayproduced by the methods of the present disclosure are described, forexample, in Bentley et al., Nature 456:53-59 (2008), WO 04/018497; U.S.Pat. No. 7,057,026; WO 91/06678; WO 07/123744; U.S. Pat. Nos. 7,329,492;7,211,414; 7,315,019; 7,405,281, and US 2008/0108082, each of which isincorporated herein by reference in its entirety.

In some embodiments of the above-described method, which employ a flowcell, only a single type of nucleotide is present in the flow cellduring a single flow step. In such embodiments, the nucleotide can beselected from the group consisting of dATP, dCTP, dGTP, dTTP, andanalogs thereof. In other embodiments of the above-described methodwhich employ a flow cell, a plurality different types of nucleotides arepresent in the flow cell during a single flow step. In such methods, thenucleotides can be selected from dATP, dCTP, dGTP, dTTP, and analogsthereof.

Determination of the nucleotide or nucleotides incorporated during eachflow step for one or more of the polynucleotides attached to the polymercoating on the surface of the substrate present in the flow cell isachieved by detecting a signal produced at or near the polynucleotidetemplate. In some embodiments of the above-described methods, thedetectable signal comprises an optical signal. In other embodiments, thedetectable signal comprises a non-optical signal. In such embodiments,the non-optical signal comprises a change in pH at or near one or moreof the polynucleotide templates.

Applications and uses of substrates of the present disclosure have beenexemplified herein with regard to nucleic acids. However, it will beunderstood that other analytes can be attached to a substrate set forthherein and analyzed. One or more analytes can be present in or on asubstrate of the present disclosure. The substrates of the presentdisclosure are particularly useful for detection of analytes, or forcarrying out synthetic reactions with analytes. Thus, any of a varietyof analytes that are to be detected, characterized, modified,synthesized, or the like can be present in or on a substrate set forthherein. Exemplary analytes include, but are not limited to, nucleicacids (e.g., DNA, RNA or analogs thereof), proteins, polysaccharides,cells, antibodies, epitopes, receptors, ligands, enzymes (e.g., kinases,phosphatases or polymerases), small molecule drug candidates, or thelike. A substrate can include multiple different species from a libraryof analytes. For example, the species can be different antibodies froman antibody library, nucleic acids having different sequences from alibrary of nucleic acids, proteins having different structure and/orfunction from a library of proteins, drug candidates from acombinatorial library of small molecules, etc.

In some embodiments, analytes can be distributed to features on asubstrate such that they are individually resolvable. For example, asingle molecule of each analyte can be present at each feature.Alternatively, analytes can be present as colonies or populations suchthat individual molecules are not necessarily resolved. The colonies orpopulations can be homogenous with respect to containing only a singlespecies of analyte (albeit in multiple copies). Taking nucleic acids asan example, each feature on a substrate can include a colony orpopulation of nucleic acids and every nucleic acid in the colony orpopulation can have the same nucleotide sequence (either single strandedor double stranded). Such colonies can be created by clusteramplification or bridge amplification as set forth previously herein.Multiple repeats of a target sequence can be present in a single nucleicacid molecule, such as a concatamer created using a rolling circleamplification procedure. Thus, a feature on a substrate can containmultiple copies of a single species of an analyte. Alternatively, acolony or population of analytes that are at a feature can include twoor more different species. For example, one or more wells on a substratecan each contain a mixed colony having two or more different nucleicacid species (i.e. nucleic acid molecules with different sequences). Thetwo or more nucleic acid species in a mixed colony can be present innon-negligible amounts, for example, allowing more than one nucleic acidto be detected in the mixed colony.

EXAMPLES

Additional embodiments are disclosed in further detail in the followingexamples, which are not in any way intended to limit the scope of theclaims.

Example 1

FIG. 5 is a workflow diagram of an example process for preparing apatterned surface using CYTOP-S that was conducted. As described above,the oxygen plasma treatment of CYTOP-S surface was found to result inloss of the hydrophobicity and chemical inertness of the CYTOP layer. Itwas discovered that certain photoresists, such as Shipley 18 seriesphotoresists, can be directly spun on the CYTOP-S coated surface withoutrequiring any oxygen plasma treatment.

Surface Preparation: First, the substrate (glass substrate or silicondioxide coated Si substrate) was cleaned with isopropyl alcohol (IPA),deionized water (DI) and then blown dry with nitrogen gas. Then, thesubstrate was placed in a vacuum desiccator at 60° C. for 12 hours forsilanization with APTMS. Then, 0.5% CYTOP-A coating solution wasspun-coated on the substrate surface at 2000 rpm for 20 seconds. Thecoated substrate was soft-baked at 50° C. for 30 min. Subsequently, 5%CYTOP-S coating solution was spun-coated on to CYTOP-A layer at 1000 rpmfor 30 seconds. To prepare the CYTOP-A and CYTOP-S solutions, afluorocarbon-based solvent such as the CT-SOLV180 from AGC was used. Thesubstrate was dried at room temperature for 30 min, and then baked at50° C. for 30 min, followed by 80° C. for 30 min, and finally baked at250° C. for 30 min. The CYTOP-A/S coating was complete and the substratewas ready for photolithography.

Photolithography: Shipley S1800 photoresist was directly spun coatedover the CYTOP layer of the substrate surface (e.g., 3000 rpm for 30seconds to coat Shipley S1805 with a thickness of 0.5 μm). The substratewas soft-baked at 115° C. for 60 seconds. Either contact aligners orsteppers with G-line UV may be used for photolithography with exposureenergy around 120 mJ/cm². The development process was conducted byputting the substrates into Microposit MF-321 developer for 60 sec, thenrinsing with deionized water followed by nitrogen gas blow dry. TheShipley S18 photoresist patterned substrate was then hard-baked in 120°C. oven for 30 min. To etch away the CYTOP in the well area to exposethe underneath SiO₂ surface, the substrate was treated with O₂ plasma(Parallel Plate Plasma Etcher, 100 sccm O₂ flux, 150 W, 180 sec dryetch). The resulting surface of the substrate comprised patterned,exposed SiO₂ surface separated by interstitial regions covered byCYTOP-S, ready for the hydrogel patterning steps.

Hydrogel Patterning: a silane coupling agent was deposited on thetreated surface of the substrate, covering both the exposed SiO₂ surfaceand the interstitial regions covered by CYTOP-S. Then hydrogel wasspun-coated over the silane coupling agent to form covalent bonding suchthat the hydrogel was immobilized on the surface. After curing, theexcess hydrogel was rinsed away. The hydrogel patterned surface can bedirectly used in oligo grafting without polishing.

FIGS. 6A and 6B are fluorescent images of a patterned device surfacecontaining 14 μm microwells with 28 μm pitch before and after clusteringand 14 cycle sequencing. The device surface was prepared according tothe Shipley photoresist workflow described in FIG. 5 using Shipley 51805with a total thickness of 370 nm for the combined CYTOP-A and CYTOP-Slayer. FIG. 6A illustrates the image of a PAZAM patterned surfacegrafted with oligo primers labeled with TET dye. FIG. 6B illustrates thegrowth of DNA clusters in microwells labeled with SYTOX® intercalatingdye. It is clear from these images that there is no sign of non-specificbinding of primer or DNA clusters in the hydrophobic CYTOP-Sinterstitial regions between the microwells.

The process was replicated on devices containing 700 nm microwells with1.8 μm pitch that were fabricated using the same Shipley 18 photoresistworkflow described in FIG. 5. FIG. 6C is a fluorescent image ofpatterned DNA clusters in 700 nm microwells visualized by SYTOX®intercalator dye. Again, the image showed very clean CYTOP-Sinterstitial regions.

Example 2

In this example, two processes of creating a patterned surface usingstandard photoresist without the need of oxygen plasma treatment of thesurface prior to photolithography were carried out—a direct patterningprocess and a lift-off process.

FIG. 7 illustrates a direct patterning workflow for creating a patternedsurface.

Surface Preparation: First, CYTOP-A and CYTOP-S were coated onto to asurface of a solid support following the same procedure as described inExample 1. Then, a fluorosurfactant Suflon-5651 was mixed withisopropanol (IPA) to form 1% solution and spun-coated over the CYTOP-Ssurface at 500 rpm for 5 seconds and then 4000 rpm for 50 seconds. Thisstep reduced the mismatch in surface energy between the CYTOP layer andthe photoresist layer to be deposited above it. Subsequently, LOR resistfrom MICROCHEM was directly spun over the treated surface withoutrequiring any oxygen plasma treatment. This approach expands the processworkflow to enable the use of a large variety of photoresists that areused in the fabrication facilities beyond Shipley 18 photoresists. Thesubsequent steps in this workflow include photoresist coating,soft-baking, UV alignment/exposure, and developing as appropriate for agiven photoresist product. To etch away the CYTOP polymer in the wellregions to expose the underneath SiO₂ area, the substrate was treatedwith O₂ plasma (Parallel Plate Plasma Etcher, 100 sccm O₂ flux, 150 W,180 sec dry etch). The resulting surface of the substrate comprisedpatterned exposed SiO₂ surface separated by hydrophobic interstitialregions, ready for the hydrogel patterning steps.

Direct patterning: First, the photoresist remaining on the substratesurface was removed by sonicating the substrate in acetone for 10 min,following with IPA rinse, water rinse, and then air blow dry.Alternatively, photoresist LOR may be stripped by MICROCHEM Remover PGand then Suflon-S651 may be stripped by acetone. The removal of thephotoresist and the fluorosurfactant exposed the underlying CYTOP-Scoating as the interstitial regions. Subsequently, the substrate wasplaced in a vacuum desiccator at 60° C. for 12 hours for norbornenesilanization. After the silanization was complete, the substrate wasthen coated with PAZAM and incubated in 60° C. oven for 1 hour. Theexcess hydrogel was rinsed away with DI water. The substrate was thensonicated in DI water at 45° C. for 30 min to remove the excess hydrogelthat loosely remained on the surface without covalent bonding. Theresulting substrate will have hydrogel coated in the well area and cleanCYTOP interstitial area free of hydrogel. The substrate is ready for thefollowing primer grafting and DNA seeding and sequencing.

FIG. 8A illustrates a lift-off patterning workflow for creating apatterned surface. The substrate fabrication process was the same asthat described above in the workflow exemplified in FIG. 7. After CYTOPand Surflon coating and photolithography, the patterned surface wasetched to expose the underlying SiO₂ surface in the wells. Then, thepatterned substrate with photoresist layer remaining on the surface wasdirectly put in a vacuum desiccator at 60° C. for 12 hours fornorbornene silanization. The substrate was then coated with PAZAM andincubated in 60° C. oven for 1 hour. The excess hydrogel was rinsed awaywith DI water. The substrate was then sonicated in DI water at 45° C.for 30 min to remove hydrogel that loosely remained on the surfacewithout covalent bonding. Subsequently, the substrate was sonicated inacetone at 45° C. for 30 min to remove the photoresist layer at theinterstitial regions. The hydrogel deposited on top of the photoresistlayer was also removed at the same time. The clean CYTOP surface at theinterstitial regions was exposed. The resulting substrate surface hashydrogel immobilized in the well areas and clean CYTOP interstitialregions free of hydrogel. The substrate is then ready for the followingprimer grafting and DNA seeding and sequencing.

FIG. 8B illustrates a fluorescent image of patterned PAZAM and DNAclusters in 14 μm microwell structures using the lift-off workflowexemplified in FIG. 8A. DNA clusters were stained with SYTOX®Intercalator dye. The image suggests that the hydrogel patterning resultis comparable to that achieved with the directing patterning workflow inFIG. 7.

In addition, the CYTOP-S surface resumed surface hydrophobicity afterthe hydrogel patterning in both direct and lift-off processes, with thelift-off method retaining better surface hydrophobicity compared to thedirect patterning method.

Example 3

Patterned flow cells with a CYTOP A surface and various well patternswere prepared as described herein. Amplification of DNA sequences wasperformed using ExAmp amplification methods and 2×150 cycle run.Incubation was run for 1 min, and 15 sec for deblocking, and reactionswere run at 65 uL volumes. The following results were obtained, andresults are shown in FIGS. 13A-13C.

Well Cluster PF Phas/Prephas Aligned Error Rate Size (%) (%) % ≥ Q30Intensity (%) 100 cycle (%) 0.7 um 59 0.117/0.162 97.68 375 ± 0 99.39 ±0.00 0.53 ± 0.00 (0.052/0.121) 0.9 um 49 0.094/0.115 96.78 345 ± 0 99.33± 0.00 0.56 ± 0.00 (0.225/0.250) 1.1 um 37 0.097/0.111 96.88 374 ± 099.43 ± 0.00 0.50 ± 0.00 0.358/0.350

The results demonstrate that CYTOP patterning is compatiable withIllumina SBS chemistry, and that the surface is robust to allowthousance of flow exchanges to complete 2×150 bps sequencing runs.

What is claimed is:
 1. A method of preparing a patterned surface withgel-coated contours, comprising: providing a solid support having asurface covered by a continuous hydrophobic coating layer, wherein thehydrophobic coating layer is in direct contact with the surface or thehydrophobic coating layer is in contact with the surface via a firstadhesion promoting layer; disposing a photoresist layer on thehydrophobic coating layer of the solid support; developing thephotoresist layer to form micro-scale or nano-scale contours on thesurface separated by hydrophobic interstitial regions; removing theremaining photoresist layer; and depositing a layer of a gel materialwithin the micro-scale or nano-scale contours, wherein the gel materialis capable of covalently bonding to oligonucleotides.
 2. The method ofclaim 1, further comprising etching off the hydrophobic coating layer inat least a portion of the micro-scale or nano-scale contours prior todepositing the gel material.
 3. The method of claim 2, furthercomprising applying a binding material layer or a silane layer to thesurface to cover at least a portion of the micro-scale or nano-scalecontours free of the hydrophobic coating layer prior to depositing thelayer of the gel material.
 4. The method of claim 3, further comprisingremoving excess gel material such that the hydrophobic interstitialregions are substantially free of the gel material.
 5. The method ofclaim 3, wherein the binding material or the silane layer comprises anorbornene derivatized silane.
 6. The method of claim 1, wherein thehydrophobic coating layer comprises a fluorinated polymer, aperfluorinated polymer, or a silicon polymer, or a mixture thereof. 7.The method of claim 6, wherein the hydrophobic coating layer comprisesan amorphous fluoropolymer, an amorphous fluoropolymer having a backbonestructure

and end functional groups selected from the group consisting ofcarboxyl, silylated amide, and trifluoromethyl, apolytetrafluoroethylene, parylen, a fluorinated hydrocarbon, afluoroacrylic copolymer, a fluorosilane, a plasma-depositedfluorocarbon, a silicon polymer, a polydimethylsiloxane, or a siloxane,or a mixture thereof.
 8. The method of claim 1, wherein the firstadhesion promoting layer comprises an amorphous fluoropolymer having abackbone structure

and carboxy end functional groups, (3-aminopropyl)trimethoxysilane(APTMS), or (3-aminopropyl)triethyoxysilane (APTES), or a combinationthereof.
 9. The method of claim 1, wherein the gel material comprisespoly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM).
 10. Amethod for preparing a patterned surface for analytic applications,comprising: providing a solid support having a surface covered by acontinuous hydrophobic coating layer, wherein the hydrophobic coatinglayer is in direct contact with the surface or the hydrophobic coatinglayer is in contact with the surface via a first adhesion promotinglayer; disposing a photoresist layer and a second adhesion promotinglayer on the hydrophobic coating layer of the solid support, wherein thephotoresist layer is contact with the hydrophobic coating layer via thesecond adhesion promoting layer; developing the photoresist layer toform micro-scale or nano-scale contours on the surface separated byhydrophobic interstitial regions; etching off the hydrophobic coatinglayer in at least a portion of the micro-scale or nano-scale contours;applying a binding material layer or a silane layer to the surface tocover at least a portion of the micro-scale or nano-scale contours freeof hydrophobic coating layer; and covalently attaching a gel material tothe binding material layer or silane layer.
 11. The method of claim 10,further comprising removing the remaining photoresist layer and thesecond adhesion promoting layer prior to applying the bonding materiallayer or the silane layer to the surface.
 12. The method of claim 10,further comprising removing the remaining photoresist layer and thesecond adhesion promoting layer after covalently attached the gelmaterial to the binding material layer or silane layer.
 13. The methodof claim 10, further comprising removing excess gel material such thatthe hydrophobic interstitial regions are substantially free of the gelmaterial.
 14. The method of claim 10, wherein the binding material orthe silane layer comprises a norbornene derivatized silane.
 15. Themethod of claim 10, wherein the hydrophobic coating layer comprises anamorphous fluoropolymer, an amorphous fluoropolymer having a backbonestructure

and end functional groups selected from the group consisting ofcarboxyl, silylated amide, and trifluoromethyl, apolytetrafluoroethylene, parylen, a fluorinated hydrocarbon, afluoroacrylic copolymer, a fluorosilane, a plasma-depositedfluorocarbon, a silicon polymer, a polydimethylsiloxane, or a siloxane,or a mixture thereof.
 16. The method of claim 10, wherein the firstadhesion promoting layer comprises an amorphous fluoropolymer having abackbone structure

and carboxy end functional groups, (3-aminopropyl)trimethoxysilane(APTMS), or (3-aminopropyl)triethyoxysilane (APTES), or a combinationthereof.
 17. The method of claim 10, wherein the second adhesionpromoting layer comprises one or more fluorinated surfactants.
 18. Themethod of claim 10, wherein the gel material comprisespoly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM).
 19. Amethod of preparing an array of polynucleotides, comprising providing asolid support comprising a patterned surface comprising microscale ornanoscale contours coated with a gel material capable of covalentlybonding to oligonucleotides, the patterned surface is prepared by themethod of claim 10; and covalently attaching a plurality of firstoligonucleotides and a plurality of second oligonucleotides to the gelmaterial.
 20. The method of claim 19, further comprising contacting theplurality of first oligonucleotides attached to the polymer coating withtemplates to be amplified, each template comprising at the 3′ end asequence capable of hybridizing to the first oligonucleotides and at the5′ end a sequence the complement of which is capable of hybridizing tothe second oligonucleotides.
 21. The method of claim 20, furthercomprising amplifying the templates using the first oligonucleotides andthe second oligonucleotides, thereby generating a clustered array ofpolynucleotides.